The muscarinic acetylcholine (ACh) receptors (mAChRs) are prototypic members of the superfamily of class I GPCRs. Accumulating evidence suggests that GPCRs do not function in isolation but are part of an intricate network of receptor-protein interactions that are responsible for various functions such as trafficking and targeting of the receptor to the membrane surface, stabilization of the receptor at the cell surface, and fine-tuning of the receptors pharmacology. Many of newly identified GPCR-interacting proteins may represent targets for novel therapeutic agents.[unreadable] Studies with M3 receptor mutant mice suggest that drugs acting on the M3 mAChR may become clinically useful in the treatment of several important pathophysiological conditions including obesity and type 2 diabetes (Wess et al. Nat Rev Drug Discov 6, 721-33, 2007). At present, little is known about receptor-associated proteins (other than G proteins, receptor kinases, or arrestins) that can modulate the activity of the M3 mAChR subtype. However, such knowledge is essential for better understanding how this receptor functions at the molecular level. Moreover, the identification of M3 receptor-associated proteins may open novel therapeutic perspectives.[unreadable] [unreadable] Identification of novel M3 receptor-interacting proteins[unreadable] In the past, many GPCR-interacting proteins have been identified by the use of classical yeast two-hybrid screening techniques. Usually, this approach requires that both interacting proteins are expressed, in a soluble form, in the nucleus. As a result, these screens cannot be applied to full-length GPCRs which are localized to cellular membranes. To overcome the limitations associated with the use of traditional yeast two-hybrid screening approaches, we employed the split ubiquitin membrane-based yeast two-hybrid system (Thaminy et al., Meth Mol Biol 261, 297-312, 2004) to screen for M3 receptor-interacting proteins. The main advantage of this system, as compared to traditional yeast two-hybrid screening approaches, is that the bait protein (the full-length M3 mAChR or any other GPCR) is localized to the plasma membrane (or other cellular membranes) and proteins that are able to interact with the bait GPCR are identified by the use of simple growth and colorimetric assays. This approach has been successfully employed to identify proteins that are able to interact with other classes of membrane proteins (for experimental details see: Thaminy et al., Meth Mol Biol 261, 297-312, 2004). [unreadable] Initially, we expressed the M3 receptor in yeast where it served as a bait to screen a human brain cDNA library for M3 receptor-interacting proteins (the M3 receptor is widely expressed throughout the brain). The subsequent split ubiquitin yeast two-hybrid screen yielded >50 individual proteins that were able to interact with the M3 receptor in a specific fashion in yeast; most of these interactions have not been identified previously. We recently confirmed the specificity of several of these interactions in a mammalian expression system. Studies are presently underway to determine the functional roles of the newly identified M3 receptor-interacting proteins in mammalian cells. Among several other techniques, we are employing siRNA technology to knock down the expression of specific M3 receptor-associated proteins in a neuronal cell line expressing endogenous M3 mAChRs. These studies should reveal the role of specific M3 receptor-associated proteins in M3 mAChR function. Given the lack of ligands that can selectively activate or inhibit M3 receptors with high selectivity, we speculate that the functional characterization of these newly identified M3 receptor-associated proteins may suggest new strategies to modulate M3 receptor function for therapeutic purposes.[unreadable] [unreadable] Molecular basis of GPCR dimerization/oligomerization[unreadable] Accumulating evidence suggests that GPCRs exist on the cell surface as dimers and/or higher order oligomers. GPCR dimerization seems to be critical for proper receptor trafficking and various other aspects of receptor function. The molecular mechanisms underlying GPCR dimerization are not well understood at present. We recently initiated a project aimed at identifying regions and/or particular amino acids involved in GPCR dimerization, using the M3 mAChR as a model receptor. This line of work involves M3 receptor random mutagenesis in yeast and the use of yeast genetic screens to reveal the effect of the introduced mutations on the ability of the M3 receptor to dimerize.[unreadable] In the first step, we subjected individual transmembrane (TM) domains of the M3 receptor to saturation random mutagenesis, creating a series of mutant M3 receptor libraries. The mutant receptors contained in these libraries are currently being analyzed for their ability to dimerize in yeast using the split-ubiquitin method (see above). In this system, one version of the M3 receptor is fused to the C-terminal half of ubiquitin. A second version of the M3 receptor is fused to the N-terminal half of ubiquitin. M3 receptor dimerization leads to the reconstitution of the two halves of ubiquitin, which is then recognized by a ubiquitin-specific protease. The protease cleaves off a transcription factor that has been attached to the C-terminal half of ubiquitin which (the transcription factor) then travels to the nucleus to transcribe specific reporter genes, allowing yeast cells containing M3 receptor dimers to grow on selective media. Given the comprehensive nature of this screen, this approach should provide new insights into the structural basis underlying M3 receptor dimerization. Given the high structural homology found among most GPCRs, these findings should be of broad general relevance. We speculate that pharmacologic strategies designed to interfere with or promote GPCR dimer formation may become useful to modulate GPCR activity for therapeutic purposes.